Direct Immunogold Labeling of Aquaporin-4 in Square Arrays of Astrocyte and Ependymocyte Plasma Membranes in Rat Brain and Spinal Cord

Proc. Natl. Acad. Sci. USA Vol. 95, pp. 11981–11986, September 1998 Neurobiology Direct immunogold labeling of aquaporin-4 in square arrays of astrocyte and ependymocyte plasma membranes in rat brain and spinal cord i JOHN E. RASH*†‡§,THOMAS YASUMURA*, C. SUE HUDSON*, PETER AGRE¶, AND SØREN NIELSEN *Department of Anatomy and Neurobiology, †Program in Molecular, Cellular, and Integrative Neurosciences, and ‡Program in Cell and Molecular Biology, Colorado State University, Fort Collins, CO 80523; ¶Departments of Biological Chemistry and Medicine, Johns Hopkins University School of Medicine; and iDepartment of Cell Biology, Institute of Anatomy, University of Aarhus, Aarhus, Denmark Communicated by Thomas S. Reese, National Institutes of Health, Bethesda, MD, August 13, 1998 (received for review March 1, 1998) ABSTRACT Aquaporin (AQP) water channels are abun- the glia limitans and that surround capillaries (3). AQP4 is dant in the brain and spinal cord, where AQP1 and AQP4 are especially abundant in astrocytes and ependymocytes in os- believed to play major roles in water metabolism and osmo- mosensory areas, including the supraoptic nucleus and sub- regulation. Immunocytochemical analysis of the brain re- fornical organ (3). These sites suggest a role for AQP4 in water cently revealed that AQP4 has a highly polarized distribution, transport between the brain parenchyma, cerebrospinal fluid, with marked expression in astrocyte end-feet that surround and blood, as well as a role in osmoregulation. capillaries and form the glia limitans; however, the structural Naturally occurring membranes enriched in AQP1 contain organization of AQP4 has remained unknown. In freeze- intramembrane particles (IMPs). Nevertheless, membrane re- fracture replicas, astrocyte end-feet contain abundant square constitution of AQP1 at high concentration yielded highly arrays of intramembrane particles that parallel the distribu- ordered two-dimensional crystalline lattices of AQP1, with a tion of AQP4. To determine whether astrocyte and ependy- tetrameric assembly of subunits (8). The major intrinsic pro- mocyte square arrays contain AQP4, we employed immuno- tein of lens MIP26 (or AQP0) occurs in lens fibers as tetragonal gold labeling of SDS-washed freeze-fracture replicas and arrays (9). Similar “square arrays”** also are found in the renal stereoscopic confirmation of tissue binding. Antibodies to collecting-duct principal cell, where multiple AQPs are ex- AQP4 directly labeled '33% of square arrays in astrocyte and pressed (12). These precedents suggest that some AQPs reside ependymocyte plasma membranes in rat brain and spinal in native tissues as square arrays. cord. Overall, 84% of labels were present beneath square In contrast, early freeze-fracture studies of astrocyte end- arrays; 11% were beneath particle clusters that resembled feet revealed an abundance of naturally occurring square square arrays that had been altered during fixation or cleav- arrays of IMPs in the plasma membranes directly facing ing; and 5% were beneath the much larger areas of glial capillaries, as well as in the end-feet that form the glia limitans plasma membrane that were devoid of square arrays. Based on bordering the subarachnoid space (13–15). These areas cor- this evidence that AQP4 is concentrated in glial square arrays, respond to the sites were AQP4 is expressed (3). The possibility freeze-fracture methods may now provide biophysical insights that square arrays in the brain and spinal cord are formed from regarding neuropathological states in which abnormal fluid AQP4 was also suggested by the apparent absence of square shifts are accompanied by alterations in the aggregation state arrays in brains from mice with AQP4 gene disruption (16). By or the molecular architecture of square arrays. using a new direct-labeling method for fracture-labeling IMPs in SDS-washed membranes (17), we sought to establish Water transport is important in multiple physiological pro- whether AQP4 proteins (i) are organized within square arrays cesses of the brain and spinal cord, including secretion and in astrocytes and ependymocytes from rat brain and spinal absorption of cerebrospinal fluid, movement of fluid across cord, (ii) exist primarily as dispersed IMPs outside of square the blood–brain barrier, osmosensation, and regulation of arrays, or (iii) occur in both clustered and dispersed IMP renal water conservation. Precise control of cell volume is distributions. critical, because the brain is encased within the rigid cranium, and thus, even minor changes in water metabolism may result MATERIALS AND METHODS in fatal compressive cerebral edema. In other settings, brain swelling may produce neonatal hydrocephalus, failure of syn- Antibodies and Immunogold Labels. For correlation with aptic transmission, or altered neuronal excitability (1–3). Ab- data from previous reports (3), affinity-purified antibodies to normal osmoregulation also may contribute to the pathogen- rat AQP4 were generated against a synthetic peptide corre- esis of epilepsy, stroke, and trauma to the brain and spinal cord sponding to amino acids 280–296 (LL182; see ref. 18), a (4, 5). domain predicted to lie in the middle of the 71-residue Molecular pathways for the movement of water across cytoplasmic C terminus (19). Monoclonal antibodies to gap- biological membranes were unknown until the discovery of the junction connexin-43 (Cx43) and polyclonal antibodies conju- aquaporin (AQP) family of membrane water channels (4, 6). Of these, two AQPs are expressed abundantly in the mamma- Abbreviations: AQP, Aquaporin; Cx43, Connexin-43; IMP, intramem- lian brain: AQP1 in the choroid plexus of the ventricles (where brane particle. § it apparently mediates secretion of cerebrospinal fluid; see To whom reprint requests should be addressed at: Department of Anatomy and Neurobiology, Colorado State University, Fort Collins, refs. 4 and 7) and AQP4 in plasma membranes of ependymal CO 80523. e-mail: [email protected]. cells and astrocytes, particularly in astrocyte end-feet that form **These distinctive IMP arrays were originally called ‘‘rectilinear arrays’’ (10), but subsequently have been called ‘‘square arrays,’’ The publication costs of this article were defrayed in part by page charge ‘‘rectangular arrays,’’ ‘‘orthogonal arrays,’’ ‘‘orthogonal assemblies,’’ ‘‘assemblies,’’ ‘‘orthogonally arranged particles’’ (or ‘‘OAPs’’), and payment. This article must therefore be hereby marked ‘‘advertisement’’ in ‘‘tetragonally arranged particles’’ (reviewed in ref. 11). Because accordance with 18 U.S.C. §1734 solely to indicate this fact. these arrays are composed of equilateral rectangles, we employ the © 1998 by The National Academy of Sciences 0027-8424y98y9511981-6$2.00y0 term ‘‘square arrays,’’ which more accurately describes the organi- PNAS is available online at www.pnas.org. zation of their subunits. 11981 Downloaded by guest on September 23, 2021 11982 Neurobiology: Rash et al. Proc. Natl. Acad. Sci. USA 95 (1998) gated to 10- and 20-nm gold beads (goat anti-mouse and goat Analysis of Immunogold Labeling. After membrane split- anti-rabbit IgG) were obtained from Chemicon. ting and replication with platinumycarbon, vigorous washing Animals. For light-microscopic and thin-section electron- with SDS dissolves away most of the tissue. However, sufficient microscopic immunocytochemistry, rats were perfused with numbers of the replicated proteins remain adsorbed to the 0.1% glutaraldehyde and 2% formaldehyde in 0.1 M cacody- replica to permit direct immunocytochemical labeling and late, pH 7.4. For freeze-fracture immunocytochemistry, adult semiquantitative analysis (17, 24). This method is best suited Sprague–Dawley rats (two males and eight females) were fixed for studying densely packed protein arrays consisting of iden- for 10 min by perfusion with 1% formaldehyde in Sorenson’s tical epitopes. phosphate buffer (SPB), pH 7.4. We photographed 10 replicas from 10 labeling experiments, Immunoblotting. Cerebellum, spinal cord, and cerebral yielding 24 areas of astrocyte and ependymocyte plasma cortex (including hippocampus) were homogenized (0.3 M membrane totaling 24 mm2 (range: 0.1–3.5 mm2 per image). sucrosey25 mM imidazoley1 mM EDTA, pH 7.2y8.5 mM Gold labels were counted beneath (i) P-face square arrays; (ii) leupeptiny1 mM phenylmethylsulfonyl fluoride). After cen- E-face square arrays; (iii) IMP clusters on astrocyte and trifugation at 4,000 3 g for 15 min at 4°C, the supernatant was ependymocyte membrane P faces; (iv) P faces that did not centrifuged at 17,000 or 200,000 3 g for 1 h. Pellets were contain square arrays or IMP clusters; (v) surrounding areas of solubilized in Laemmli sample buffer containing 2% SDS and replicated extracellular space; and (vi) replicated plasma mem- run on 12% polyacrylamide minigels. Immunoblots were an- branes of neurons, oligodendrocytes, and vascular endothelial alyzed with affinity-purified anti-AQP4 (18) and visualized cells. Analysis of the latter two areas allowed rates of nonspe- with the Enhanced Chemiluminescence System (Amersham). cific binding to be determined. Immunocytochemistry. Fixed-tissue blocks of brain and Gold beads were defined as associated with square arrays if spinal cord were infiltrated for 30 min with 2.3 M sucrose they were located directly beneath or within 20 nm of the array containing 2% formaldehyde, mounted on holders, and rapidly (i.e., within a radius two antibody molecules in length). The frozen in liquid nitrogen. For light-microscopic

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